This invention generally relates to the field of diagnostic virology. More particularly, the invention relates to methods for culturing and assaying a virus and diagnostic kits and cell lines for doing the same.
Viruses and other microorganisms which infect humans, as well as other organisms, are numerous. Many of these viruses are associated with assorted clinical manifestations. Early detection of the viral etiological agents causing the clinical manifestation is desirable for effective treatment and management of the infected organism. Detection of even small concentrations of virus is desirable.
Tissue culture techniques using various cell lines are the most widely used means of assaying viruses. Numerous cell lines have been developed for culturing viruses. However, relatively few are useful because of the tendency of many cell lines to be unstable, to develop undesirable characteristics, or to be insensitive for viral growth. Accordingly, a more sensitive cell line for isolation and detection of viruses is a constant goal of diagnostic virologists.
Ideal cells for culturing viruses are cells which are sensitive for a variety of viral pathogens. However, such cell cultures have yet to be developed. Thus, several different cell cultures are required to diagnose various virus types. Such cell cultures are either primary cells or cell strains or lines arising from primary cells. Primary cells are started from cells taken directly from the tissue or organs of an organism. Primary cells are considered to be primary cells until subcultured for the first time wherein they are regarded as cell lines. Primary cells are expended by use so that a continuous supply of primary cells from new tissue from living donors is needed. This may require sacrifice of the donor. Furthermore, each new batch of primary cells poses a risk of endogenous viral, bacterial or fungal contamination.
Use of cell lines may minimize these problems. Cell lines arise from the first subculturing of primary cells. Such cell lines may be finite, i.e., are able to be subcultured only a limited number of times before senescence, or continuous, i.e., are able to be subcultured an infinite number of times. Such continuous cell lines are referred to as xe2x80x9cestablishedxe2x80x9d cell lines when it is demonstrated the cell line may be subcultured indefinitely in vitro.
Recently, an improved shell vial technique has been developed for the detection of viruses from clinical specimens. This method may be combined with blind immunohistochemistry staining of the inoculated culture with viral specific antibody to identify the virus. This technique is commonly used in diagnostic virology laboratories. However, the method relies on the susceptibility of the cell line, used for virus culture, to viral infection. Low susceptibility of the cell line to viral infection results in poor diagnostic sensitivity.
Another recent advancement, polymerase chain reaction (PCR) technology, has been used to detect slow growing viruses, viruses which cannot be grown in cell culture, or viruses which are difficult to recover in cell culture from a specific specimen site. An example is herpes simplex virus from cerebrospinal fluid specimens. However, this technique detects viral nucleic acid and not infectious virus. When it is possible, the detection of infectious virus is often preferred because it correlates more with clinical manifestation. Accordingly, PCR detection of viral nucleic acid may only be indicative of the presence of remnants of a past infection or the presence of a latent infection.
The effect of butyrate on mammalian cells has been discussed. Prasad, K. N. et al, (1976) xe2x80x9cEffect of Sodium Butyrate on Mammalian Cells in Culture: a Review,xe2x80x9d In Vitro 12, 125-132. The effect of sodium butyrate on various specific cell-virus interactions has also been examined. For instance between: (1) Epstein-Barr Virus/human lympho-blastoid cells, Luka, Janos et al, (1979) xe2x80x9cInduction of the Epstein-Barr Virus (EBV) Cycle in Latently Infected Cells by n-Butyrate,xe2x80x9d Virology, 94, 228-231; (2) herpes simplex virus/neuroblastoma cells, Ash, Ronald J., (1986) xe2x80x9cButyrate Induced Reversal of Herpes Simplex Virus Restriction In Neuroblastoma Cells,xe2x80x9d Virology, 155, 584-592; (3) human endothelial cells/cytomegalovirus, Radsak, K., et al, (1989) xe2x80x9cInduction by Sodium Butyrate of Cytomegalovirus Replication in Human Endothelial Cells,xe2x80x9d Arch. Virology, 107, 151-158; and (4) human epithelial cells/cytomegalovirus, Tanaka, J. et al, (1991) xe2x80x9cSodium Butyrate-Inducible Replication of Human cytomegalovirus in a Human Epithelial Cell Line,xe2x80x9d Virology, 185, 271-280.
International patent publication WO 89/06686 discloses use of butyric acid and salts thereof to enhance the efficiency of specific protein production by cultured eukaryotic cells.
This invention relates to a method of culturing a virus including the steps of: (A) providing cells from a cell line susceptible to infection by the virus and a specimen; (B) treating the cells with a compound of the formula RC(O)Q, wherein Q is R, OR, OX, or X, each R is independently hydrogen or a hydrocarbyl group containing 1 to about 10 carbon atoms and wherein X is hydrogen or a cation; (C) inoculating the treated cells with the specimen; and (D) incubating the inoculated cells to allow viral growth to proceed.
In accordance with another aspect of the invention is a diagnostic kit for assaying a virus includes (A) cells from a cell line susceptible to infection by the virus; (B) a compound of the formula RC(O)Q, wherein Q is R, OR, OX, or X, each R is independently hydrogen or a hydrocarbyl group containing 1 to about 10 carbon atoms and wherein X is hydrogen or a cation; and (C) reagents for assaying the virus.
In another aspect of the invention is a method for culturing a virus, comprising the steps of: (1) inoculating treated cells from a cell line susceptible to infection by the virus with the specimen, wherein the cells are treated with a compound of the formula RC(O)Q, wherein Q is R, OR, OX or X, each R is independently hydrogen or a hydrocarbyl group containing 1 to about 10 carbon atoms and wherein X is hydrogen or a cation; and (2) incubating the inoculated cells to allow viral growth to proceed.
In accordance with still another aspect of the invention is a method for assaying a virus, includes (A) providing cells from a cell line susceptible to infection by the virus and a specimen; (B) treating the cells with a compound of the formula RC(O)Q, wherein Q is R, OR, OX, or X, each R is independently hydrogen or a hydrocarbyl group containing 1 to about 10 carbon atoms and wherein X is hydrogen or a cation; (C) inoculating the treated cells with the specimen; (D) incubating the inoculated cells to allow viral growth to proceed; and (E) assaying the virus in the cells.
In accordance with a further aspect of the invention is an enhanced mink lung cell line having improved susceptibility to infection by a herpes virus prepared by treating mink lung cells identified as ATCC #CCL64 with a compound of the formula RC(O)Q, wherein Q is R, OR, OX or X, each R is independently hydrogen or a hydrocarbyl group containing 1 to about 10 carbon atoms and wherein X is hydrogen or a cation.
In another aspect of the invention, the method relates to a diagnostic kit for assaying a virus, comprising: (1) treated cells from a cell line susceptible to infection by the virus, wherein the cells have been treated with a compound of the formula RC(O)Q, wherein Q is R, OR, OX or X, each R is independently hydrogen or a hydrocarbyl group containing 1 to about 10 carbon atoms and wherein X is hydrogen or a cation; and (2) reagents for assaying the virus.
In another aspect of the invention, the method relates a method for assaying a virus, comprising the steps of: (1) inoculating treated cells from a cell line susceptible to infection by the virus, wherein the cells have been treated with a compound of the formula RC(O)Q, wherein Q is R, OR, OX or X, each R is independently hydrogen or a hydrocarbyl group containing 1 to about 10 carbon atoms and wherein X is hydrogen or a cation; and treated cells with the specimen; (2) incubating the inoculated cells to allow viral growth to proceed; and (3) assaying the virus in the cells.
The claimed methods, diagnostic kits and enhanced cell lines provide and have improved sensitivity for virus assay and enhanced susceptibility to virus infection.